PROJECT SUMMARY Around 46% of the World's population lives in risk of contracting a mosquito-borne disease. Our overall goal is to produce mosquito pathogens that can effectively suppress vector borne diseases, and to this end we have engineered the mosquito pathogenic fungus Metarhizium pingshaense (Mp) to specifically deliver insecticidal proteins into mosquito blood. These genetically modified pathogens achieve fast kill at low spore dosages to block malaria transmission. The present challenge is to convert this promising strategy into a validated public health intervention by resolving outstanding issues related to the release of genetically modified organisms. Host specificity is a critical issue in any form of biological control, and a common critique is that introduced biocontrol agents may evolve to attack non target species. The likelihood of such evolution depends on the number of genes involved, how they interact, and the time scale at which host shift events happen. There is currently a dearth of information overall on determinants of host range for most pathogen-host systems, even though specificity and host shifts are of great concern for human, plant and animal health. Fortuitously, mosquito pathogenic Mp strains are an ideal model system as they have close relations that are broad spectrum generalist pathogens, while other lines have specialized to other insects (beetles, other flies etc). To preemptively identify and address concerns about the possibility of mosquito pathogen host shifts, we propose to compare genomes of 14 generalist and specialist strains and analyze correlations between gene divergence and host acceptance. This will: 1) provide the first characterization of the armamentarium of mosquito specific Mp strains; 2) greatly increase our knowledge of the genetics of host specificity; 3) define time lines for host shifts and the appropriate evolutionary distances for addressing various risks; 4) provide a much needed model for how new diseases originate, and 5) provide a timely new approach to risk assessment of biological agents (genetically modified or not) that accounts for past evolutionary host range changes when predicting future evolutionary changes. 4) enable scientists from a developing country to become directly involved in evaluating the potential use and application of transgenic microbes for future disease control.